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Because of its versatility (all types of substrates), robustness (Z′ > 0.7), rapid performance (10 minutes), and ease of use, the luminescence-based Kinase Glo® Assay platform has gained wide acceptance in many drug screening programs for protein kinase inhibitors. The uniqueness of this assay lies in its use of ATP depletion as a readout for kinase activity, which is a general feature of all kinases, and thus it is applicable to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, protein, polymer, lipids). For example, we have used it to monitor the activity of MAPK using peptide as well as protein substrate (MBP), the activity of PI3 kinase using phospholipids substrate, and the activity of glucokinase using glucose as substrate. It also detects additional phosphorylation sites of already existing phosphopeptide substrates by enzymes such as GSK-3 and CK1 and monitors the activity of kinases phosphorylating their substrates on multiple sites. Like other luciferase-based assays, the maximum linear range of ATP concentration used is limited by the linearity of the luminescence reactions, and it saturates at about 10–15μM. We show here that the maximum limit of ATP concentration can be extended to 100μM. This will make it feasible to screen libraries for compounds that are non ATP competitors, which broadens the selection of inhibitors. We show data with serine/threonine protein kinases as well as tyrosine protein kinases. We have used the Kinase Glo® Assay to screen for the best kinase substrate for kinases and thus providing valuable information on those library compounds using the most optimal substrate for the enzyme at significantly high ATP concentrations. The output luminescence of Kinase Glo® Plus is intensely bright, and the signal is stable for hours. Finally, the assay is robust (as indicated by the high Z′ values [over 0.7]), homogenous, can be completed in one step after completion of kinase reaction, does not require antibodies or custom synthesized substrates, and is the most inexpensive to screen for optimal substrates of a peptide library as well as for ATP and non-ATP competitive inhibitors.

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